Thymoquinone attenuates diabetes-induced hepatic damage in rat via regulation of oxidative/nitrosative stress, apoptosis, and inflammatory cascade with molecular docking approach

Diabetes mellitus (DM) is a complex metabolic condition that causes organ dysfunction. The current experiment sought to determine the effect of thymoquinone (TQ) on hyperglycemia, hyperlipidemia, oxidative/nitrosative stress, inflammation, and apoptosis in diabetic rats prompted by streptozotocin (STZ) (55 mg/kg body weight i/p). The animals were allocated into control, TQ (50 mg/kg B.W. orally administered for 4 succeeding weeks), Diabetic, and Diabetic + TQ groups. This study confirmed that TQ preserves the levels of insulin, fasting blood glucose, HOMA β-cell indices, HbA1c %, body weight, and lipid profile substantially relative to the DC group. Furthermore, hepatic antioxidant (CAT, GSH, and T-SOD) values were reduced. Conversely, the enzymatic activity of liver functions (AST, ALT, ALP, cytochrome P450, and hepatic glucose-6-phosphatase), lipid peroxidation (MDA), pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), nitric oxide (NO) and inflammatory marker (CRP) enhanced with STZ administration, which is substantially restored after TQ treatment. Relative to the diabetic rats, TQ reestablished the hepatic architectural changes and collagen fibers. Additionally, TQ downregulated the intensity of the immunohistochemical staining of pro-apoptotic marker (caspase-3), p53, and tumor necrosis factor-alpha (TNF-α) proteins in hepatic tissues. Furthermore, TQ displayed abilities to interact and inhibit the binding site of caspase-3, interleukin-6 receptor, interleukin-1 receptor type 1, TNF receptor superfamily member 1A, and TNF receptor superfamily member 1B in rats following the molecular docking modeling. All these data re-establish the liver functions, antioxidant enzymes, anti-inflammatory markers, and anti-apoptotic proteins impacts of TQ in STZ-induced DM rats. Founded on these outcomes, the experiment proposes that TQ is a novel natural supplement with various clinical applications, including managing DM, which in turn is recommended to play a pivotal role in preventing the progression of diabetes mellitus.


Experimental design
Rats were allocated to 4 groups at random, each with eight rats: i. Rats in group 1 (control group) were administrated with 1 mL of distilled water / 100 g of BW/day through gastric gavage.ii.Rats in group 2 (TQ group) were administered with TQ, firstly dissolved by the addition of dimethyl sulfoxide, then by adding normal saline (for a final dimethyl sulfoxide value of less than 0.5%).Subsequent TQ solution was given at 50 mg/kg of BW one time per day by oral gavage for up to four weeks 8 .The dosage was accustomed weekly following any alteration in body weight to sustain a parallel dose per kg BW of rats over the whole experimental period for every group.iii.Rats in group 3 (Diabetic) were rendered diabetic as previously described and were administered 1 mL of distilled water / 100 g of BW/day by gastric gavage.iv.Rats in group 4 (Diabetic + TQ) were rendered diabetic and administrated with TQ as defined with group 2.

Body weight measurements
The body weights of all animals were measured at the start of each week, and findings were analyzed accordingly.

Oral glucose tolerance test (OGTT)
After ending the experiment, animals were deprived overnight and then given glucose solution (2 g of glucose/ 5ml distilled water/kg Bwt of rats) intragastrically.Samples of the blood were drawn from the tail vein at 0, 30, 60, 90, and 120 min.Glucose levels were evaluated immediately in the blood samples by Hemoglucotest® glucose strips 20,24,26 .

Blood and tissue assembly and preparation
Twenty-four hours following the last treatment administration, with ketamine/xylazine anesthesia (7.5 and 1.0 mg/kg via intra-peritoneal infusion) 23 , 2 samples of the blood were collected from the heart on sodium fluoride at room temperature.One sample was utilized to calculate HbA1c%, and the other was centrifuged at 1,800 × g for 15 min to separate plasma for insulin, glucose, liver function tests, lipid profile, and inflammatory marker analysis.
Vol:.( 1234567890) www.nature.com/scientificreports/Following blood collection, and while the animals were still anesthetized, euthanasia was performed via decapitation, and dissected, each liver excised and thoroughly estimated.A part of the hepatic tissue of every rat was washed with deionized water and physiological saline solution (NaCl 0.9%) to remove RBCs and platelets; tissues were blotted with blotting paper and perfused with 50 mM sodium phosphate saline buffer (100 mM Na 2 HPO 4 /NaH 2 PO 4 , pH 7.4) in an ice-cold medium with 0.1 mM EDTA.Then, tissues were shredded in 10 mL of ice-cold buffer/g tissue and centrifuged for 30 min at 10,000 × g.The supernatant was transferred to Eppendorf tubes and stored at -80°C until oxidative/nitrosative and anti-oxidative enzyme activities were determined.Another section of liver tissue was removed and promptly preserved in 10% buffered formaldehyde for histological and immunohistochemical analysis.

Homeostasis model assessment of β-cell function (HOMA-β-cell) index
For HOMA β-cell index determination, plasma insulin, and fasting glucose levels were utilized according to Matthews's formula 29 : Insulin sensitivity indices: fasting glucose/insulin ratio and insulin-1 were calculated from plasma insulin and fasting glucose concentrations 30 .

Liver function tests and lipid profile measurements
ALT (Cat#: DD0440), AST (Cat#: DD0453), and ALP (Cat#: DD0363) content were estimated using commercially available kits (Diamond Diagnostics Co., Cairo, Egypt) as per the manufacturer's directions.The activity of plasma's cytochrome p450 (CYP450) (Cat#: BD34701) was determined through ELISA kits.Markers of lipid profile, including TAG, TC, HDL-C, and LDL-C, were analyzed by kits (Bio-Diagnostics Co., Cairo, Egypt), and the data were interpreted consequently.The atherogenic index in plasma (AIP) was determined through the formula = log (TAG/HDL-C) 22 .

Histopathological examination and semi-quantitative lesion scoring
Hepatic tissue samples were obtained from 8 rats per group and promptly maintained in buffered formalin 10% for a minimum of 24 h.Hepatic samples were washed, dehydrated with serial alcohol dilutions, cleaned in xylene, and inserted in paraffin at 60°C.To assess the extent of collagen, paraffin slices of five microns thickness were produced and stained with hematoxylin and eosin (H and E) and Masson's trichrome stain, then inspected beneath a light microscope.The extent of hepatic tissue damage was evaluated using a semiquantitative scoring assay, in which five random fields were examined from each section.The severity of lesions was scored and graded according to the percentage of affected tissue, as follows: none (-) = 0%, representing no involvement of the examined field; mild ( +) = 5-25% of the examined field; moderate (+ +) = 25-50% of the examined field; and severe (+ + +) ≥ 50-100% of the examined field 37 .

Immunohistochemical evaluation
Following the manufacturer's directions, a standard horseradish peroxidase-immunohistochemical approach employing rabbit anti-rat p53, caspase-3, and TNF-α was smeared to positively charged slides of paraffin Sects 38 .Five-micron slices of hepatic tissue were dewaxed, rehydrated, and incubated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity.The slides were antigen-retrieved by putting them in a microwave oven for 10 min in a 10 mM sodium citrate buffer (pH 6.0).Then, endogenous peroxidase activity was blocked with 3% H 2 O 2 for 10 min.Nonspecific proteins were inhibited by 2% bovine serum albumin.The slices were rinsed www.nature.com/scientificreports/three times in Dako Tris-buffered saline before being incubated overnight at 4°C with a primary rabbit polyclonal anti-p53 antibody (1:100) (PA5-32,045; Thermo-Fisher Scientific, WA, USA), rabbit polyclonal anti-caspase-3 antibody (1:100) (Code# ab4051; Abcam, Cambridge, UK) and rabbit polyclonal anti-TNF-α antibody (1:100) (PA5-41,057; Thermo-Fisher Scientific, WA, USA).The tissue slices were rinsed in Tris-buffered saline and then incubated with the streptavidin-horseradish peroxidase reagent and a biotinylated secondary antibody for 30 min at 37°C.Slides were then treated with a 3, 3' diaminobenzidine substrate chromogen solution, counterstained with Mayer's hematoxylin, and photographed.Images of 10 different fields, at a magnification of × 400, were analyzed using ImageJ software to estimate the area (%) of caspase3, P-53 and TNF-α positive brown immunostained cells 37 .

Ligand preparation
The three-dimensional (3D) structure of TQ was retrieved from the PubChem (https:// pubch em.ncbi.nlm.nih.gov/) database in SDF format and opened in MOE 2015.10 39 software for energy minimization and docking with target proteins.

Molecular docking analysis and visualization
Target proteins were docked with TQ using MOE software and the protein-ligand interactions were visualized by the same software.

Statistical analysis
GraphPad Prism v.9 (https:// www.graph pad.com/) (GraphPad, San Diego, CA, USA) analyzed the data by a one-way ANOVA with Tukey's post hoc multiple range testing.Two-way ANOVA analyzed body weight and OGTT data with Tukey's post hoc multiple range testing.P < 0.05 was required for all significance declarations.

Body weights
Weights of the body of the STZ-induced diabetic animals decreased markedly (P < 0.001) than those of the control non-diabetic group in the 3rd and 4th weeks of the study.However, TQ-treated diabetic rats improved the weights of the body (P < 0.001) relative to the diabetic non-treated animals in the 3rd and 4th weeks of the study.A nonsignificant variation was documented between the weights of control non-diabetic and TQ-treated rats (Fig. 1).

Oral glucose tolerance test
Differences in blood glucose levels of all groups throughout various time courses (0-120 min) are displayed in Fig. 2. The maximum value of glucose was identified after 30 min of receiving glucose (2 g/kg B.W.) and then reduced to main levels within 2 h in all experimental animals except STZ-diabetic control, which displayed an extreme glucose intolerance (P < 0.001) throughout the entire time course (0-120 min).Nonetheless, diabetic animals treated with TQ had substantially (P < 0.001) lower glucose levels than the normal control group.During the various time courses of the oral glucose tolerance test, there was no significant variation in blood glucose concentrations of the control non-diabetic and TQ-treated rats.

Blood glycemic parameters and β-cell function indices
As revealed in Fig. 3, compared to non-diabetic control animals, there were statistically marked (P < 0.001) increases in fasting plasma glucose (Fig. 3A), glucose/insulin ratio (Fig. 3C), HbA1c% (Fig. 3F) and insulin −1 (Fig. 3D) in the diabetic group.Acquired data also displayed that there was a substantial reduction in HOMAβcell index (Fig. 3E) and fasting insulin (Fig. 3B) value in STZ-diabetic rats as relative to non-diabetic control animals.Those parameters improved significantly in diabetic TQ-treated rats relative to diabetic control rats, with fasting plasma glucose, insulin −1 , HbA1c%, and glucose/insulin ratio significantly (P < 0.001) lowered in diabetic animals treated with TQ relative to the diabetic group.While the HOMA-cell index and fasting insulin were markedly (P < 0.001) higher in the Diabetic + TQ treated animals relative to the Diabetic group.

Hepatic function tests and lipid profile
Our findings revealed that STZ-induced diabetes induced a marked (P < 0.001) enhancement in plasma ALT (Fig. 4A), AST (Fig. 4B), and ALP (Fig. 4C) values, which indicates a hepatic tissue injury and destruction of the hepatocyte cell membrane.Moreover, it also enhanced plasma CYP450 (Fig. 4D).TQ therapy of diabetic rats reduced these effects substantially (P < 0.001) reducing hepatic function tests and CYP450 activity relative to diabetic control rats.The hepatic function tests and CYP450 activity were not markedly variant between the TQ-treated group and the control non-diabetic one.Figure 5 displays the lipid profile and atherogenic index in normal and experimental rats in each group.STZinduced diabetes triggered a marked (P < 0.001) enhancement in plasma TC (Fig. 5A), TAG (Fig. 5B), LDL-C (Fig. 5D), and atherogenic index (Fig. 5E) values with a marked reduction in HDL-C (Fig. 5C).Treating diabetic animals with TQ led to a marked (P < 0.001) down-regulation in plasma TC, LDL-C, TAG, and atherogenic index values relative to the diabetic group.TQ enhanced the marked reduction in HDL-C of diabetic animals.A non-significant difference was recorded between the control non-diabetic and TQ-treated rats considering the

Pro-inflammatory cytokines and inflammatory marker
STZ-induced diabetic control rats displayed a marked (P < 0.001) enhancement in plasma concentrations of TNF-α (Fig. 6A), IL-1β (Fig. 6B), IL-6 (Fig. 6C), and CRP (Fig. 6D) relative to the normal control group, which indicate the higher grade of the inflammatory cascade in the liver through diabetes.Nevertheless, TQ administration reduced markedly (P < 0.001) degree of enhanced proinflammatory, and inflammatory markers relative to diabetic control rats.A non-significant variation was documented in proinflammatory, and inflammatory biomarkers between the control non-diabetic and TQ-treated rats.

Oxidant-antioxidant status and G6Pase
The diabetic group showed a substantial (P < 0.001) enhancement in hepatic MDA (lipid peroxidation marker) (Fig. 7A), nitrite (Fig. 7B) values, and the activity of hepatic G6Pase (Fig. 7F); which is linked with a marked reduction in GSH (Fig. 7C) value and T-SOD (Fig. 7D) and CAT (Fig. 7E) activities relative to the normal control group.On the contrary, TQ treatment of diabetic rats markedly (P < 0.001) reduced the degree of increased LPO, nitrosative markers, and G6Pase hepatic activity relative to the diabetic control animals.Additionally, relative to diabetic rats, the antioxidant markers were mostly recovered in the Diabetic + TQ animals.TQ alone administrated rats revealed a marked (P < 0.01) reduction in intensity of MDA and nitrite concentrations relative to non-diabetic control animals.Furthermore, the antioxidant enzymatic activities in the TQ-treated rats were considerably (P < 0.01) enhanced than in normal control animals.Regarding the hepatic G6Pase activity, there was a non-marked variation between the TQ-treated group and the control non-diabetic one.

Histopathological findings
No histological variations were observed between non-diabetic control and TQ-administrated rats in the liver.So, they were deliberated as control.Control and TQ-treated rats' hepatic tissue displayed a normal histological structure, with undamaged hepatic architecture in hepatic lobules, portal areas, and hepatic vasculature (Fig. 8A).meanwhile, hepatocytes of the diabetic group exhibited diffuse congestion of hepatic vasculature (hepatic sinusoids and central vein) (Table 1 and Fig. 8B), diffuse hepatic hydropic degeneration where the cells were swollen,  1 and Fig. 8C) alongside hepatocellular necrosis with mononuclear cell infiltration (Table 1 and Fig. 8D) and intense mononuclear cell infiltrations in the portal area (Table 1 and Fig. 8E).Simultaneously, hepatic tissue sections of the Diabetic + TQ group displayed nearly normal histoarchitecture (Table 1 and Fig. 8F).
The obvious lesions of Masson's trichrome in control and TQ-treated rats were fine light green collagen fibers (Fig. 9A and B).Diabetic rats displayed moderate and extensive thick collagen fibers around the central vein, in the portal area, and hepatic parenchyma (Fig. 9C, D, and E), while the Diabetic + TQ rats showed fine collagen fibers (Fig. 9F).

Immunohistochemistry
Nuclear and cytoplasmic immunostaining of hepatocytes of the non-diabetic control and TQ-administrated groups displayed a negative staining for the expression of caspase-3 protein (Fig. 10A and B).Conversely, diabetic animals exhibited moderate to strong positive brown immune reactions for caspase-3 protein (Fig. 10C).At the same time, Diabetic + TQ rats were weak immune brown staining for caspase-3 protein (Fig. 10D).
The immunohistochemical estimation of p53 protein of the hepatocytes of the control and TQ-treated animals displayed negative brown staining (Fig. 10E and F).On the contrary, diabetic rats exhibited strong to moderate nuclear-positive brown immunoreactivity of p53 protein expression (Fig. 10G).Nonetheless, there was weak nuclear immune brown staining in Diabetic + TQ rats (Fig. 10H).
The hepatic tissue immunostaining of the inflammatory TNF-α protein in control and TQ-administrated animals displayed a negative brown staining (Fig. 10I and J).In contrast, the diabetic group revealed moderate to strong positive brown reactions for TNF-α protein (Fig. 10K).In the Diabetic + TQ rats, there was less dense and weak brown staining (Fig. 10L).

Molecular docking assessment
Molecular docking interactions and scores of TQ against caspase-3, IL-6R, IL1R1, TNFRSF1A, and TNFRSF1B   binding sites are illustrated in Fig. 11.TQ interacted with the binding site of caspase-3 with a binding energy of -4.94 kcal/mol by H-acceptor with ARG207 and H-pi with TRP206 residues (Fig. 11A).Figure 11B explores the interaction of TQ with the IL-6R binding site by -4.68 kcal/mol, binding energy.Furthermore, TQ interacted with GLY423 (pi-H) residue in the IL1R1 binding site by binding energy of -4.90 kcal/mol (Fig. 11C).By three H-acceptor bonds (GLN111, HIS140, and PHE141), TQ interacted with TNFRSF1A's binding site by binding energy of -4.55 kcal/mol (Fig. 11D).Although TQ interacted with TNFRSF1B's binding site by binding energy of -4.36 kcal/mol with GLY169 (H-acceptor) residue.

Discussion
In many metabolic dysfunctions, antioxidant use is seen as an approach to reestablishing normal physiological balance 19 .DM is a systemic, metabolic, and endocrine illness defined mostly by hyperglycemia.It is also linked to glucose, lipid, and protein metabolism changes.This is because antioxidant processes within the body are being reduced and impaired 22,25 .Thymoquinone (TQ), the primary ingredient of N. sativa, is thought to be a hypoglycemic and anti-oxidative substance that might offset the side effects and enhanced cost of pharmaceutical medications 3,41 .The liver is the tissue utmost vulnerable to the effects of hyperglycemia-induced oxidative stress.Different processes, such as inflammation and oxidative stress, promote hepatocyte damage 20 .STZ is a diabetogenic and hepatotoxic substance that destroys the cell membrane of pancreatic beta-cells, breaks DNA, and interacts with many enzymes, counting glucokinase, causing insulin levels to drop dramatically 23 .It is critical to investigate the impact of anti-diabetic drugs in STZ-triggered hyperglycemia in research models.TQ was administered to animals in this experiment to evaluate their anti-diabetic and hepato-protective capabilities.
The current study displayed that STZ considerably reduced body weights in the third and fourth weeks of the experiment.The results are congruent with those of Almatroodi et al 42 .and Abdelrazek et al 22 .This might be due to decreased glucose and amino acid availability to cells of the body, which results in a deficiency of substrates required for the biosynthesis of the cells and can disrupt connected cellular metabolism, resulting in muscular wastage 2 .TQ treatment for diabetic rats caused a marked enhancement in the weight of the body relative to the diabetic animals.Present findings follow those of Abdelrazek et al 22 .and Abdel-Moneim et al 43 .This could support TQ's anabolic/antidiabetic action on diabetes-induced muscle atrophy due to glucose inaccessibility 16,42 .
Diabetic rats had lower blood insulin and HOMA β-cell function indicators than control rats.In contrast, the diabetes group had higher plasma glucose levels, HbA1c%, and glucose/insulin ratio than the control group.These data are consistent with those of Hafez et al 20 and Faisal Lutfi et al 8 .HbA1c% is a widely used biomarker for assessing the severity of diabetes and its consequences 23 .This impact is caused by STZ's alkylating toxic activity on pancreatic islet β-cells, which limits insulin secretion, resulting in hyperglycemia 19 .TQ caused a significant drop and increase in plasma glucose and insulin levels compared to diabetic control rats.These findings agreed with those of Hofni et al 2 .and Abdelrazek et al 22 .TQ's plasma glucose lowering impact may demonstrate its insulinotropic activity, where it might cause partial regeneration of pancreatic islet β-cells, resulting in increased insulin synthesis and peripheral use 16 .TQ, in addition to its capacity to reduce intestinal glucose absorption, has an inhibiting impact on the expression of gluconeogenic enzymes and glucose synthesis in the liver 16,22 .Furthermore, it has the ability to stimulate adenosine monophosphate-activated protein kinase in the liver and muscles, blocking gluconeogenesis 42 .TQ lowered blood glucose during the OGTT, which might be attributed to TQ's capacity to stimulate pancreatic β-cells insulin production 16 .Another factor is quinine's hypoglycemic feature, part of the TQ molecule's structure 41 .The mechanisms of action might be via inhibiting gluconeogenesis in hepatic tissue or glucose uptake www.nature.com/scientificreports/into adipose tissues and muscles or by increasing glucose consumption in tissues 42 .As a result, unlike insulin and other synthetic medicines, TQ may be presumed to have anti-hyperglycemic properties 22,43 without causing any undesirable hypoglycemia.STZ promotes experimental hyperglycemia in rats, increasing ALT, AST, ALP, CYP450, and hepatic G6Pase activity, which is a strong sign of liver injury.AST and ALT are closely related to converting amino acids to keto acids and are known to rise with DM 43 .CYP450 isoforms are a class of enzymes that can catalyze various enzymatic processes, including xenobiotic metabolism 17,44 .G6Pase is a critical and rate-limiting enzyme in gluconeogenesis, as demonstrated by an experiment in which G6Pase knockout mice had symptoms such as hyperlipidemia and lactic acid buildup 45 .The elevated activities of serum markers of liver function (ALP, ALT, AST, CYP450, and G6Pase) in the current investigation suggested that hepatic damage could be caused by the generation of hyperglycemia by infusing STZ.The rise in AST and ALT activity could be attributed to impairment of the cells in the hepatic tissue due to STZ-induced diabetes 20 .The present experiment revealed that treating diabetic rats with TQ reduces liver function enzyme activity to virtually normal levels, indicating that TQ protects liver cellular functioning.Previous research has shown that TQ positively affects hepatic key enzymes in diabetic rats 3,41 .It was also previously revealed that insulin has a role in downregulating CYP450 and G6Pase 22 , which could explain why TQ therapy enhanced insulin levels in diabetic rats.
According to the present experiment, within the diabetic control group, STZ caused dyslipidemia (lower HDL-C and higher TC, LDL-C, TAG, and AIP).These results agreed with those of Abdelrazek et al 22 .DMinduced dyslipidemia develops as a result of hyperglycemia and insulin deficiency, which enhances lipolysis and fatty acid production from adipose tissue to the circulation with changes in the metabolism, raising LDL-C, TC, and TAG values and predisposing to cardiovascular dysfunction 43 .TQ administration improves the diabetesinduced aberrant lipid profile, which is in line with the results of Abdelrazek et al 22 .and Abdel-Moneim et al 43 .TQ's beneficial effects on DM-induced dyslipidemia might be attributed to its stimulation of the activity of hepatic arylesterase, antioxidant properties, and adjusting impacts on the genes influencing cholesterol metabolism 1,3 .
Lipid peroxidation is a symptom of cellular impairment caused by ROS.In addition to MDA generation, excessive concentrations of ROS promote lipid peroxidation of the cellular membrane 2,46 .The true procedure of STZ-administrated DM in animals implicates ROS formation, DNA alkylation, and an increase in NO production in β-cells of the pancreas.Decrease of β-cell mass and function is the primary cause of diabetes and initiates dysfunction of the pancreatic β-cell 22 .Diabetes-related oxidative/nitrosative stress causes glucose autoxidation, lipid peroxidation, protein glycation, and decreased activity of antioxidant enzymes 1 .Lipid peroxidation might be produced by oxidative stress with enhanced mitochondrial uncoupling protein expression, activating cytokines, ligands, and growth factors, resulting in fibrosis and inflammation 47 .MDA and nitrite levels in the hepatic tissue were considerably greater in STZ-administrated diabetic animals.TQ administration also markedly reduced hepatic oxidative/nitrosative stress indicators, suggesting that TQ possess an important influence as a hepatic protective agent in contradiction to STZ diabetic mice, principally through its antioxidant characteristics 2,42 .
The present experiment considered the effect of TQ on oxidative stress of the liver in STZ DM in male rats.Fundamental processes of diabetes-induced free radicals and oxidative stress have been widely studied 19,20 .Diabetic rats had considerably lower hepatic GSH levels, activities of T-SOD, and CAT because of hyperglycemia and hyperlipidemia, which reduce the natural antioxidant activities and stimulate the generation of free radicals 3,8 .TQ treatment increased the antioxidant reserve of GSH levels as well as T-SOD and CAT activities in hepatic tissues, supporting the findings of Almatroodi et al 42 .,Abdelrazek et al 22 ., and Abdel-Moneim et al 26 .The inclusion of quinine in the TQ structure confirms its antioxidant capabilities.Quinine improves the admission to cellular and subcellular components, facilitating ROS removal 16 .TQ reduced oxidative stress by inhibiting nonenzymatic lipid peroxidation 2,41 .Furthermore, TQ's hypoglycemic impact enhances its antioxidant effect by regulating hyperglycemia-induced ROS.
These findings were confirmed in our work by immunostaining for the hepatic tissue inflammatory TNF-α protein of diabetic rats, which demonstrated a moderate to strong positive brown reaction for TNF-α protein with a marked increase in the immunohistochemical staining of TNF-α.Inflammation plays a significant role in the progression of diabetes and is consequently related to increased resistance to insulin and decreased responsiveness in the target tissues of insulin 18 .Furthermore, pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) and inflammatory markers (CRP) are important markers of pancreatic β-cell dysfunction and insulin resistance, all of which provide the DM progression 3,42 .These findings were confirmed in our work by immunostaining for the inflammatory protein of TNF-α in the liver of diabetic rats, which demonstrated a moderate to strong positive brown reaction for TNF-α protein.Circulating values of most proinflammatory cytokines are often raised in DM, owing to hyperglycemia, which raises cytokine levels in the blood via an oxidative mechanism 48 .It was discovered that diabetic liver had considerably enhanced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) protein production and the inflammatory infiltration of macrophages 1 .TNF-α was significantly elevated in the STZ diabetic group, with enhancing DNA destruction in the hepatic tissue, which also substantially enhanced the stimulation of nuclear factor kappa B, Janus kinases-signal transducer and stimulator of transcription, and c-Jun N-terminal kinases signaling pathways 16 .TQ treatment reduced liver TNF-α protein levels and immunostaining significantly.According to our findings, STZ may trigger an inflammatory mechanism that contributes to diabetic liver damage 20 .Previous research has shown that TQ decreases pro-inflammatory markers (IL-6, TNF-α, and cyclooxygenase-2) 18,49 .Because of its anti-inflammatory capabilities, TQ suppressed the overproduction of pro-inflammatory markers (IL-6, IL-1β, and TNF-α), an inflammatory indicator (CRP), and immunostaining of TNF-α in STZ-administrated diabetic rats 2,41 .These findings were also validated by the current study's molecular docking findings.TQ displayed affinity to interact with and block the binding sites of IL-6R, IL1R1, TNFRSF1A, and TNFRSF1B, which may explain TQ's anti-inflammatory effects, as recognized by Alyami and Al-Hariri 16 .
Microscopic examination of normal control and TQ hepatic tissue revealed normal histological structure, with undamaged hepatic architecture in hepatic lobules, portal regions, and hepatic vasculature.STZ-induced diabetic rats had diffuse hepatic vasculature congestion, diffuse hepatic hydropic degeneration, and intense mononuclear cell infiltrations in the portal area, which was accompanied by a moderate or extensive thick collagen fiber around the portal vein and hepatic parenchyma 50 .STZ has a degenerative effect on hepatocytes, in addition to free radical buildup and damage due to oxidative stress 45 .The pathophysiology of diabetic hepatic injury may be due to inadequate insulin production or insulin resistance caused by DM, which causes hyperglycemia and hyperlipidemia due to carbohydrate metabolism disruption 47 .Diabetic rats given TQ had essentially normal histoarchitecture.Because of its antioxidant, anti-inflammatory, and hypoglycemic properties, TQ is a promising candidate for managing diabetes complications 16 .
Apoptosis, or programmed cell death, is a physiological mechanism of the body that performs a critical function in maintaining the body's environmental stability.It is a biomarker that responds to persistent stress.Excessive or insufficient apoptosis would be harmful to the body.It is also considered an important defensive mechanism because it regulates many effector cells 20 .A pro-apoptotic marker (caspase-3) is an effector protein in the intrinsic and extrinsic apoptotic mechanisms 47 .The immunohistochemical and quantitative analyses of our experiment revealed that the STZ-diabetic group had considerably higher caspase-3 immunopositive hepatic cell counts.Faisal Lutfi et al 8 .discovered that STZ-induced oxidative damage in male diabetic rats' hepatic tissues indicated a high presence of caspase-3 positive cells and a low expression of Bcl2.TQ reduced the expression of caspase-3 and p53 proteins in the hepatic tissue of diabetic rats, implying that TQ protects the liver by inhibiting apoptosis and inflammation 41 ; this was also confirmed by the findings of the current study's molecular docking, which revealed that TQ interacts with and inhibits the caspase-3 binding site.
ROS causes DNA damage and stimulates a response to it, leading to the overexpression of p53.Chronic hyperglycemia followed by an increase in cytoplasmic glucose concentration causes C-terminal glycosylation of the tumor suppressor p53, which then activates the transcriptional activator p53, resulting in its translocation to the nucleus and the initiation of transcription of several p53-dependent genes 21,51 .The overall response of many cell types to DNA-damaging chemicals is typically characterized by p53 protein overexpression 52 .The tumor suppressor gene p53 governs cell cycle progression, induces apoptosis, and is integrated in progression of diabetic complications in response to DNA damage at the G1/S checkpoint 51 .Diabetes patients had higher levels of p53 in their livers, which was associated with the resistance to insulin 21 .Because TQ modulates blood glucose concentrations in diabetic animals, TQ treatment lowered immunostaining of p53, which could indicate that TQ has an anti-apoptotic effect in the handling of DM 2,42 .

Conclusion
To conclude, TQ has anti-diabetic, anti-inflammatory, anti-apoptotic and hepatic protective actions via modulating blood glucose and lipid profile, as well as enhancing liver functioning, hepatic histoarchitecture and oxidative stress status.In the current work, we found that TQ displayed abilities to interact and inhibit the binding site of caspase-3, interleukin-6 receptor, interleukin-1 receptor type 1 and TNF receptor in rats following the molecular docking modeling.Overall, TQ alleviates most of the alterations seen in diabetic animals and effectively ameliorates the harm caused by STZ; consequently, it's recommended to be integrated with the diabetes mellites management protocols.TQ's detailed molecular mechanism in diabetes management must be further investigated.

Figure 1 .
Figure 1.Assessment of body weight.Data were analyzed with a two-way ANOVA followed by Tukey's multiple comparison test.Data are expressed as the mean ± SD. n = 8. ns = nonsignificant, *** P ˂ 0.001, and **** P ˂ 0.0001.

Figure 2 .
Figure 2. Assessment of glucose tolerance test.Data were analyzed with a two-way ANOVA followed by Tukey's multiple comparison test.Data are expressed as the mean ± SD. n = 8. **** P ˂ 0.0001.

Figure 8 .
Figure 8. Photomicrograph of a rat liver section stained by H and E × 400.(a) Control rats showing normal histology with normal central vein (CV) and hepatocytes (b, c, d, e) diabetic rats showing congestion of hepatic sinusoid (long black arrows) and central vein (short black arrows), diffuse hydropic degeneration of hepatocytes (arrowheads) beside hepatocellular necrosis with mononuclear cell infiltrations (black star) and intense mononuclear cell infiltrations in portal area (white star) (f) TQ + diabetic treated rats showing normal hepatic histoarchitecture with normal central vein (CV).(Scale bar = 50 μm).